Dopamine transporter (DAT) is a transmembrane protein expressed in dopaminergic neurons where it is necessary for reuptake of extracellular dopamine (DA) back into DA neurons leading to termination of DA neurotransmission. Since the main role of DAT is DA clearance, DAT is vital for numerous physiological functions involving DA neurotransmission such as cognition, locomotion, motivation and reward-related behaviors. On the other hand, DAT is also linked to several neurological disorders with abnormal DA neurotransmission such as Parkinson's disease, schizophrenia, attention deficit hyperactivity disorder and drug addiction. DAT participates in DA clearance only when it is present on the plasma membrane. The levels of DAT at the plasma membrane are tightly regulated by the process of endocytosis but mechanisms of DAT endocytic trafficking are poorly understood. Most of the current knowledge on regulation of DAT trafficking is based on studies done in non-neuronal expression systems and therefore the mechanism of DAT trafficking in DA neurons remains unclear. A better comprehension of molecular mechanisms of DAT endocytic trafficking is not only crucial for understanding normal DA neurotransmission but is also important for designing strategies for prevention and treatment of abnormal DA signaling in neurological disorders. Recently, we have developed knock-in mice that express functional DAT with a HA-tag (HA-DAT). The HA-tag provides us with a novel tool to monitor HA-DAT in live DA neurons to study the process of endocytosis. In DA neurons of these mice, we found that HA-DAT undergoes constitutive endocytosis. Our preliminary studies in these mice also strongly suggest a direct interaction between DAT and an 1-subunit of G protein, Go. We found reciprocal co-immunoprecipitation and co-localization of DAT and Go in DA neurons of the knock-in mice. Additionally, inhibition of Go by pertussis toxin increased endocytosis of DAT in DA neurons of these mice. G proteins like Go are known to be the central mediators of signaling in the brain. Absence of Go in mice results in severe neurological abnormalities but the mechanisms are poorly understood. Interaction between DAT and Go is a novel finding and further studies will provide insights into the importance of these two vital proteins in normal function of DA neurons. Based on our data, we hypothesize that DAT is a direct downstream effector of Go whereby Go directly interacts with DAT and modulates its endocytic trafficking. The overall goal of this project is to employ HA-DAT mice to define molecular mechanisms of Go-regulated DAT trafficking and to identify the factors necessary for interaction between these two proteins in DA neurons. Psychostimulant drugs like amphetamine are known to modulate DA neurotransmission by regulating DAT trafficking but the molecular mechanisms are unknown. In the long run, we hope to investigate the role of Go- mediated DAT trafficking in action of amphetamine and gain insights into the pathology of drug addiction. PUBLIC HEALTH RELEVANCE: Dopamine transporter (DAT) is a protein found in the dopaminergic neurons of the brain where is it responsible for re-uptake of the neurotransmitter dopamine (DA) from extracellular space back into the dopaminergic neurons causing termination of DA neurotransmission. Abnormalities in DAT function and hence DA re-uptake leads to neurological disorders like Alzheimer's, schizophrenia, attention deficit hyperactivity disorder and drug addiction. A thorough understanding of the underlying molecular mechanisms of DAT trafficking which dictates the amount of DA re-uptake is crucial for designing strategies for prevention and treatment of neurological disorders.